Resistance of uveal melanoma to the interstrand cross-linking agent mitomycin C is associated with reduced expression of CYP450R.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular tumour of adults, frequently metastasising to the liver. Hepatic metastases are difficult to treat and are mainly unresponsive to chemotherapy. To investigate why UM are so chemo-resistant we explored the effect of interstrand cross-linking agents mitomycin C (MMC) and cisplatin in comparison with hydroxyurea (HU). METHODS: Sensitivity to MMC, cisplatin and HU was tested in established UM cell lines using clonogenic assays. The response of UM to MMC was confirmed in MTT assays using short-term cultures of primary UM. The expression of cytochrome P450 reductase (CYP450R) was analysed by western blotting, and DNA cross-linking was assessed using COMET analysis supported by γ-H2AX foci formation. RESULTS: Both established cell lines and primary cultures of UM were resistant to the cross-linking agent MMC (in each case P<0.001 in Student's t-test compared with controls). In two established UM cell lines, DNA cross-link damage was not induced by MMC (in both cases P<0.05 in Students's t-test compared with damage induced in controls). In all, 6 out of 6 UMs tested displayed reduced expression of the metabolising enzyme CYP450R and transient expression of CYP450R increased MMC sensitivity of UM. CONCLUSION: We suggest that reduced expression of CYP450R is responsible for MMC resistance of UM, through a lack of bioactivation, which can be reversed by complementing UM cell lines with CYP450R.

False diagnosis of papilloedema and idiopathic intracranial hypertension.

The diagnosis of idiopathic intracranial hypertension (IIH) relies heavily on the appearance of the optic disc. We report eighteen children referred to us over a 3 year period with disc swelling and suspected IIH. Following a tertiary ophthalmological review, papilloedema was excluded in ten with buried drusen, disc crowding, pseudopapilloedema, or misinterpretation of normal appearances. In these ten children, five had a mean opening pressure on lumbar puncture of 27.2 cm H2O, range 19-32, which was significantly lower than those with IIH (37.5 cm H2O, range 29-47; p<0.01). We conclude that diagnosis of IIH is difficult, and that more precisely defined criteria for assessment and diagnosis are needed.

Fresh frozen amniotic membrane for conjunctival reconstruction after excision of neoplastic and presumed neoplastic conjunctival lesions.

PurposeSuspicious neoplastic conjunctival lesions often require wide excision with tumour-free margins, leaving significant conjunctival defects requiring reconstruction. In this study we report the results of using fresh frozen amniotic membrane grafts (AMG) after wide excision of potentially malignant lesions.MethodsRetrospective review of 53 patients; between January 2011 and April 2014. Conjunctival lesions were excised with a non-touch technique (2 mm margin) and sent for histopathological analysis. The surgical margins were treated with cryotherapy and a fresh frozen AMG was used to cover the defect. The main features examined were for any signs of recurrence, the conjunctivalisation of the AMG, complications and cosmetic appearance.ResultsFifty-three patients; 35 males and 18 females. Mean age was 54.9 (range 19-88). The mean follow up to January 2015 for all lesions was 21.4 months (range 8-48 months). The most common lesions were invasive malignant melanoma. There were no local surgical complications in 77.3% of patients; minimal scarring (11.3%), symblepharon (11.3%), and granuloma (7.5%). Five patients with conjunctival melanoma developed in-transit metastasis and orbital extension, none of it was at the site of the AMG.ConclusionOur case series is the largest reported to date, with the largest number of melanomas. The use of fresh frozen AMG has improved the local surgical outcomes by improving healing and reducing scarring as well as allowing for a wider surgical margin.

Ultrasound biomicroscopy in the management of melanocytoma of the ciliary body with extrascleral extension.

AIM: To demonstrate the ultrasound biomicroscopic features of a ciliary body melanocytoma with extrascleral extension, and a conservative approach in its management. METHOD: Observational case reports. Two cases of ciliary body melanocytoma were suspected at presentation, confirmed histologically by biopsy, and subsequently monitored for change by serial ultrasound biomicroscopic imaging. The main outcome measures were anatomical and functional preservation of the eye, with avoidance of formal surgical excision. RESULTS: Ultrasound biomicroscopy allows clear visualisation of the tumours, and the ultrasound characteristic is of low homogeneous internal reflectivity. 5 year follow up with observation only demonstrates success with this conservative management approach. Histopathological evaluation confirmed melanocytoma. CONCLUSIONS: Melanocytoma is a rare tumour. However if considered in the differential diagnosis at presentation and confirmed histologically, further management with use of the ultrasound biomicroscope as an accurate mode of imaging is an acceptable technique for preservation of the eye and avoids surgical excision.

Regression of invasive conjunctival squamous carcinoma in an HIV-positive patient on antiretroviral therapy.

Case history of an African woman presenting with advanced HIV and a painful conjunctival lesion is presented. A conjunctival biopsy revealed invasive squamous cell carcinoma, with orbital invasion on computed tomography scan. She was commenced on antiretroviral therapy. She refused surgery to remove the eye and orbital contents (exenteration), and was referred to palliative care. Gradually, her immune status and ocular symptoms improved. At ophthalmic review, the tumour had apparently completely regressed. This unprecedented phenomenon may be due to antiretroviral therapy. Discussion covers conjunctival carcinoma and behaviour of HIV-related tumours with antiretroviral therapy. Antiretroviral drugs may offer a better alternative to disfiguring surgery in the future.

Outcomes of treatment with stereotactic radiosurgery or proton beam therapy for choroidal melanoma.

AIM: To present our experience of the use of stereotactic radiosurgery and proton beam therapy to treat posterior uveal melanoma over a 10 year period. METHODS AND MATERIALS: Case notes of patients treated with stereotactic radiosurgery (SRS), or Proton beam therapy (PBT) for posterior uveal melanoma were reviewed. Data collected included visual acuity at presentation and final review, local control rates, globe retention and complications. We analysed post-operative visual outcomes and if visual outcomes varied with proximity to the optic nerve or fovea. RESULTS: 191 patients were included in the study; 85 and 106 patients received Stereotactic radiosurgery and Proton beam therapy, respectively. Mean follow up period was 39 months in the SRS group and 34 months in the PBT group. Both treatments achieved excellent local control rates with eye retention in 98% of the SRS group and 95% in the PBT group. The stereotactic radiosurgery group showed a poorer visual prognosis with 65% losing more than 3 lines of Snellen acuity compared to 45% in the PBT group. 33% of the SRS group and 54% of proton beam patients had a visual acuity of 6/60 or better. CONCLUSIONS: Stereotactic radiosurgery and proton beam therapy are effective treatments for larger choroidal melanomas or tumours unsuitable for plaque radiotherapy. Our results suggest that patients treated with proton beam therapy retain better vision post-operatively; however, possible confounding factors include age, tumour location and systemic co-morbidities. These factors as well as the patient's preference should be considered when deciding between these two therapies.

Failure of intravitreal bevacizumab in the treatment of choroidal metastasis.

BACKGROUND: Metastasis to choroid is the most common intraocular malignancy, arising most frequently from carcinoma of breast in women and lung in men. Recent case reports have described successful use of intravitreal bevacizumab to achieve local control of such tumours. MATERIALS AND METHODS: Five cases of choroidal metastases from varying primaries: breast, lung, and colon were treated with intravitreal bevacizumab, and tumour response observed and documented with serial photographs and B-scans. RESULTS: Four of the five tumours were seen to progress despite intravitreal bevacizumab treatment. CONCLUSIONS: Intravitreal bevacizumab as the primary treatment of choroidal metastases is not recommended and should not delay more effective alternative treatments.

Alpha-melanocyte-stimulating hormone inhibits NF-kappaB activation in human melanocytes and melanoma cells.

Alpha-melanocyte-stimulating hormone is produced by several different cell types including neural cells, endothelial cells, monocytes, and keratinocytes. A biologic role in melanocyte pigmentation is widely recognized, but more recent studies describe a part in modulating inflammatory and immune responses. The aim of the this study was to investigate the mechanism by which alpha-melanocyte-stimulating hormone antagonizes proinflammatory cytokine action. We report that alpha-melanocyte-stimulating hormone (10-9 M) was effective in opposing a tumor necrosis factor-alpha stimulated increase in NF-kappaB DNA binding activity in: (i) normal ocular melanocytes; (ii) cells cultured from ocular melanoma tumors; and (iii) two cutaneous melanoma cell lines. NF-kappaB is activated by many inflammatory mediators and controls transcription of genes required for immune and inflammatory responses. The transcription factor complex was positively identified as the p50/p65 heterodimer, recognized to have transcriptional activating potential. Maximum reduction of NF-kappaB DNA binding activity with alpha-melanocyte-stimulating hormone was detected 2 h after cellular stimulation and varied from between 53% and 18% depending on cell type. Whereas the acute inhibitory effects could be mimicked by elevating cyclic adenosine monophosphate, alpha-melanocyte-stimulating hormone was not found to have any effect on the relative level of IkappaBalpha protein expression over 24 h. These data show that alpha-melanocyte-stimulating hormone has a pronounced effect on NF-kappaB activity in melanocytes and melanoma cells, identifying a specific dimeric complex, and suggest this to be a key pathway by which immunomodulation/anti-inflammation may operate. The results may also be considered in the broader context of general inflammatory pathologies concerning cells which express alpha-melanocyte-stimulating hormone receptors and utilize the NF-kappaB signaling pathway.

Investigation of the role of signal transduction in attachment of ocular melanoma cells to matrix proteins: inhibition of attachment by calmodulin antagonists including tamoxifen.

We investigated the role of signal transduction systems in the attachment of human uveal melanoma cells to matrix proteins. Ocular melanoma cells established from primary tumours attached rapidly to all substrates examined. Preferred substrates of attachment were collagens type I, III and IV and fibronectin rather than laminin, gelatin, arginine-glycine-aspartine, vitronectin, poly-L-lysine or plastic. All cells showed rapid attachment to the preferred substrates (80% within 10 min). Manipulation of intracellular cyclic AMP or protein kinase C activity had relatively little effect on cell attachment. In contrast, attachment was significantly reduced by manipulating either intracellular calcium or calmodulin. After 15 min at 37 degrees C, the calcium ionophore ionomycin (5 microM) reduced attachment to 25%, and TMB8 (50 microM), which can reduce intracellular calcium, reduced attachment to 60%. The experimental calmodulin antagonist J8 (25 microM), a substituted naphthalene sulphonamide, reduced attachment to 40%. Similarly tamoxifen (25 microM), which has calmodulin antagonist activity in vitro, reduced attachment to 55%. Both J8 and tamoxifen inhibited cell attachment to a wide range of matrix proteins, suggesting that this effect on attachment is not dependent on the presence of specific adhesion receptors. Reduction of ocular melanoma tumour cell/matrix interactions through manipulation of intracellular calcium or calmodulin may therefore merit further investigation as a possible approach to reducing metastatic spread.

Cytokine modulation of adhesion molecule expression on human retinal pigment epithelial cells.

PURPOSE: The purpose of this study was to assess qualitatively the expression of adhesion molecules by human retinal pigment epithelium (RPE) and to study their regulation by inflammatory cytokines. These molecular events and the role played by inflammatory cytokines are important for selective lymphocyte trafficking into the eye during uveitis. METHODS: Expression of intracellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular adhesion molecule (VCAM-1) by early passage human RPE cells was assessed by flow cytometry. In addition, the regulation of the expression of these molecules by the inflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma) was determined. Reverse transcription-polymerase chain reaction (RT-PCR) was used to characterize further adhesion molecule expression. RESULTS: Flow cytometric analysis determined that ICAM-1 was constitutively expressed on RPE cell lines and that in the presence of TNF alpha, IFN gamma, and IL-1 beta, there was a median fold increase in expression of 4.4, 5.4, and 4.4, respectively. In contrast, flow cytometric analysis of ELAM-1, PECAM-1, and VCAM-1 indicated that these adhesion molecules were not constitutively expressed at the cell surface; only the expression of VCAM-1 was upregulated by the presence of cytokines. The results of RT-PCR on RPE cells indicated that mRNA for all the adhesion molecules was present constitutively in some RPE cultures. After activation with IFN gamma, TNF alpha, and IL-1 beta, RT-PCR analysis showed that the number of RPE cell lines expressing all the adhesion molecules increased. CONCLUSIONS: ICAM-1 expression is markedly upregulated by inflammatory cytokines. Although mRNA for other adhesion molecules is expressed in RPE cells and is enhanced by inflammatory cytokines, this does not necessarily reflect the cell surface protein expression. Thus, the expression of adhesion molecules by RPE cells, and the subsequent recruitment of specific leukocytes, may be determined by the local cytokine environment.

Effect of melanocyte stimulating hormone on human cultured choroidal melanocytes, uveal melanoma cells, and retinal epithelial cells.

PURPOSE: To establish methodology for the culture of human choroidal melanocytes to compare their responsiveness to melanocyte stimulating hormone (MSH) with that of their transformed melanoma counterparts and with that of the retinal epithelial cell. METHODS: Choroidal melanocytes from the choroid of eyes enucleated for the presence of malignant melanoma were cultured in MCDB 153 medium supplemented with insulin, transferrin, hydrocortisone, glutamine, nystatin, vitamin E, phorbol myristate acetate, bovine hypothalamic extract, cholera toxin, and chelexed fetal calf serum. RESULTS: High yields of pure spindle-shaped bipolar melanocytes were obtained with a doubling time of 3 to 4 days in nine consecutive eyes. Cells continued to divide after 4 months in culture. In contrast, uveal malignant melanoma cells grew rapidly in a relatively simple medium of Ham's F12:DMEM (1:1) supplemented with fetal calf serum, insulin, transferrin and glutamine. This medium was unable to support choroidal melanocytes. Choroidal melanocytes were DOPA-positive with appreciable tyrosinase activity that significantly increased with treatment with MSH. MSH also significantly altered the size, local density, and distribution of primary and mature melanosomes of ocular melanocytes. In contrast, uveal melanoma cells had a low level of tyrosinase activity and failed to respond to MSH with either an increase in enzyme activity or melanosome size. Retinal epithelial cells failed to show significant tyrosinase activity under the conditions studied or any increase in melanosome size in response to MSH. CONCLUSION: Ocular melanocytes show evidence of regulation by MSH and a range of mitogenic stimuli unlike the transformed melanoma cells, implying a loss of regulatory control in the latter.

Gelatinolytic metalloproteinase secretion patterns in ocular melanoma.

Fifteen posterior uveal melanoma cell lines were analyzed qualitatively for gelatinolytic and caseinolytic proteinase activity after one to five in vitro passages. All 15 cell lines secreted a gelatinolytic metalloproteinase, with an apparent molecular weight of 72 kD, into protein-free culture media; nine of these secreted an additional gelatinolytic metalloproteinase with an apparent molecular weight of 92 kD. Neither species had the ability to degrade casein. This approach may provide insight into the mechanisms of tumor metastasis in uveal melanoma.

Susceptibility of human ocular melanoma cells to spontaneous and interferon-augmented natural cytotoxicity.

Due to the current interest in natural killer (NK) cells as a host defence mechanism against neoplasia, we have investigated the susceptibility of short-term cultures of human ocular melanoma cells to spontaneous and interferon (IFN)-augmented natural cytotoxicity. Cultures of ocular melanoma cells were readily established and identified as bipolar and multipolar pigmented melanoma cells. In short-term cytotoxicity assays these cell lines demonstrated a weak susceptibility to spontaneous human PBL natural cytotoxicity, and increased killing was observed using IFN-augmented cytotoxic effector cells. These findings may be pertinent in relation to the role of NK cells in vivo.

Melanosis oculi. An ultrastructural study of an affected iris.

The iris of a patient with melanosis oculi was studied, using the transmission electron microscope. An unusual form of macromelanosome was found within the cytoplasm of stromal melanocytes. It is possible that these structures were formed as a result of inappropriate production of the individual subunits of the melanosome. Their presence within stromal melanocytes, but not within the cells of the posterior pigment epithelium, was in keeping with the hypothesis that melanosis oculi is a disorder of melanocytes derived from neural crest.

Lysozyme distribution in human lacrimal glands and other ocular adnexa.

In an immunohistochemical study of the human lacrimal glands and other orbital adnexa, lysozyme was found to be present in the major and accessory lacrimal glands but absent from meibomian glands and conjunctival epithelium. Almost all acinar and tubular cells in major and accessory lacrimal glands contain lysozyme, although occasional cells show diminished staining for lysozyme, probably because of secretion. Only one secretory lacrimal tubuloacinar cell type is demonstrable, although two types previously have been described. Lacrimal duct cells do not contain lysozyme. The findings of this study support the concept that the tubuloacinar cells of the main and accessory lacrimal glands are the sole source of the lysozyme secreted into tears.

Clinically important ocular reactions to systemic drug therapy.

Many systemically administered drugs produce ocular adverse effects. Fortunately, relatively few are capable of causing significant, irreversible visual impairment. It is the responsibility of every clinician when prescribing systemic therapeutic agents to be aware of potential adverse ocular reactions, to appreciate their significance, and to inform the patient of the potential risks of treatment. In instances where serious adverse reactions relate to the cumulative effects of prolonged treatment, it is the responsibility of the prescribing physician to institute appropriate methods of visual screening. In this respect, it is most important to obtain the necessary individual baseline measurements before treatment is commenced. Chloroquine retinopathy is probably the most feared of all adverse ocular reactions to systemic drug therapy. However, it occurs only rarely if the daily dosage of chloroquine does not exceed 250mg. Regular screening using automated perimetry is mandatory if prolonged therapy is contemplated. Amiodarone almost inevitably produces corneal deposits. These rarely produce symptoms, and resolve upon withdrawal of the drug. Optic neuropathy has recently been described with amiodarone. Systemic anticoagulant therapy may be associated with intraocular hemorrhage in patients with pre-existing disciform macular degeneration, and such agents should be used with caution in affected individuals. Systemic corticosteroids produce posterior subcapsular cataracts in susceptible individuals which may profoundly affect visual acuity. Although elevated intraocular pressure may also result from systemic therapy, the relationship between the pressure rise and development of glaucomatous changes remains unclear. Ethambutol may produce optic neuropathy if the daily dosage exceeds 15 mg/kg. The changes are usually reversible within a few weeks of stopping treatment. High doses of tamoxifen may produce a maculopathy with loss of visual acuity, if given for prolonged periods. The risk must be weighed against the benefits of treatment. Patients receiving more than 800 mg/day of thioridazine have developed retinopathy, which is usually reversible if detected early enough. Tricyclic antidepressants and other agents with anticholinergic properties may cause disturbances of accommodation and pupillary dilatation. The latter may rarely precipitate acute angle closure glaucoma in susceptible individuals.

Cytogenetics of iris melanomas: disparity with other uveal tract melanomas.

The chromosomal alterations of iris melanomas are poorly characterized, only one report has been detailed. Cytogenetic analysis was performed on the tumors and heparinized blood samples of three patients with iris melanomas; in one case a primary tumor and its related seedling were examined. On analysis of lymphocytes, two of the patients were found to experience a low level fragility of chromosome 9, in the region of a cutaneous melanoma susceptibility gene. All iris melanoma lesions were karyotyped. Clonal abnormalities of chromosomes 3, 5, 6, 7, 8, 9, 12, 15, 17, 18, 19, and Y were found, and in one case a large number of marker chromosomes were observed. No specific chromosomal change was common to the iris melanomas, but two cases had different abnormalities of chromosomes 5 and 18. Variations between the primary tumor and its related seedling were the acquisition of an additional chromosome 15, and a polyploid form of the cell line in the seedling. This study suggests that the most common chromosomal changes of posterior uveal melanomas are less frequent in iris melanomas. Iris melanomas also appear to experience relatively high levels of chromosomal alterations, including the formation of marker chromosomes, which is perhaps reminiscent of cutaneous melanoma.

The effect of a once-daily oral dose of nadolol on intraocular pressure in normal volunteers.

We compared the effects of once-daily oral administration of 20 mg and 40 mg of nadolol vs placebo in 40 normal volunteers in a randomized, double-masked trial. Each of 23 women and 17 men (ranging in age from 18 to 50 years) had intraocular pressure, pulse rate, blood pressure, pupil size, amplitude of accommodation, and near point measured immediately before treatment and then at 24 hours, day 8, and day 15 after treatment. Both dosages of nadolol produced significant decreases in intraocular pressure at three hours (P less than .001). Although both dosages decreased intraocular pressure at 24 hours compared with placebo, only the change with the 40-mg dose was significant (P less than .05). Although both dosages produced significant decreases in pulse rate and blood pressure, side effects were minimal.

Oncocytomas (oxyphil adenomas) of the lacrimal caruncle.

Three cases of oncocytoma (oxyphil adenoma) of the lacrimal caruncle are reported. Transmission electron microscopy performed on one of the lesions confirms the tumour to be composed of cells containing abnormally large numbers of mitochondria. It is suggested that these tumours arise from accessory lacrimal glands or their secretory ducts.

Use of bovine pericardium as a wrapping material for hydroxyapatite orbital implants.

AIM: To present the results of 27 patients who had enucleation for malignant melanoma of the choroid with hydroxyapatite implant wrapped in bovine pericardium. METHOD: A retrospective study was performed on 27 patients, 12 males and 15 females, who had enucleation as a primary treatment for their choroidal melanomas. The patients were followed up at 1 week, 1 month, 3 months, and then every 6 months. A conformer was fitted at 1 week and an artificial eye at 1 month. The average follow up was 1.7 years. RESULTS: No patient had extrusion of the implant. One patient needed repair of the wound, two patients required a lateral tarsal strip, and one patient developed a conjunctival granuloma, which did not need excision. In one patient there was shallowing of the inferior fornix. The cosmetic results and ocular movement were satisfactory in all but one patient. CONCLUSION: Use of bovine pericardium as wrapping material for the hydroxyapatite implants has shown promising results with minimal extrusion rates providing an effective alternative for sclera, eliminating the potential risks of CJD.